We have identified and cultured human CNS derived progenitor cells from the human brain. These cells are nestin positive and do not express any other phenotypic marker of differentiation. Upon treatment of such cells with specific factors, these cells can be directed toward lineage pathways that make these cells neurons, astrocytes or oligodendrocytes. We are in the process of studying the genomics of this differentiation and the proteins that are uniquely made during the course of such differentiation. We use viral infection to mark phenotypes of these cells and are able to increase infection or diminish infection depending upon the pathway that these cells are directed toward. We have recently made gene expression vectors that are only expressed in specific cell lineage pathways using reporter genes for either green or red flourescent proteins. These gene products are visually recognized using video microscopy so they mark the pathway of cell differentiation. Such experiments will ultimately provide valuable insights into the nature of development of the brain and allow a key experimental tool for a large array of studies for cell therapy. We have also developed the tools to differentiate progenitor cells to oligodendrocytes, myelin basic protein producing cells with a phenotype similar to mature oligodendrocytes. However, upon infection with JCV that targets these cells in the adult brain, the differentiated oligodendrocytes are not susceptible to infection. These cells are missing the NF-1X protein transcription factor so that infection does not proceed. In addition to observations for NF-1X investigation, we have identified an inhibitor of viral expression, NF-1A, expressed at levels that block the upregulation of JCV by NF-1X. These two DNA binding proteins have similar DNA binding sites at their N terminal end for the viral DNA but differ in their C terminal ends that are transactivation regions that recognize host cell factors. The laboratory has recently had an instructional video made as part of its publication in the Journal of Visual Experiments that details the methodology developed to differentiate the mulitpotential brain derived progenitor cells through lineages for oligodendrocytes. In addition to this approach, we have more recently applied gene expression microarrays at various time points during differentiation of progenitor cells to astrocytes. Preliminary evidence shows that clusters of genes get upregulated quickly when the progenitor cells are exposed to selective medium driving differentiation to GFAP positive cells. Some of these genes are those involved in acquiring the characteristics of susceptibility to JCV infection. These observations make the link between phenotypes of cell seen in clinical brain tissue and cell models of infection in culture systems. We have furhter defined a protocol using flow cytometry to select population of cells and sort based on stages of differentiation. For example, some cells will display phenotypes based on staining patterns of cell surface markers that allow sorting in the FACS Aria II instrument following infection by viruses. Identification cells in the human developing brain then allows determination of not only stages of differentiation but also combined with microarry analysis dissects molecular levels of cell gene expression using viral tools.